1. Field of the Invention
The present invention relates generally to the fields of immunology and protein chemistry. More specifically, the present invention relates to the isolation, purification and characterization of a novel antiproliferative protein from Bacillus thuringiensis var. thuringiensis, as well as the effects of this protein on macrophage activation.
2. Description of the Related Art
Agents that display antiproliferative properties selectively against tumor cells have potential as anticancer drugs. These types of agents are sought from both synthetic as well as natural sources. Such compounds with antiproliferative properties could be either proteinaceous or nonproteinaceous in nature.
Both gram-negative and gram-positive bacteria are known to synthesize proteins that are toxic to eukaryotic cells. Bacillus thuringiensis is a gram positive bacteria that during sporulation synthesizes large quantities of a protein that kills insects when ingested (Hofte and Whiteley, 1989). This protein has been used as a microbial pesticide for more than 30 years, and it is considered harmless to humans (Green et al, 1990). Insecticidal proteins are assembled in the bacteria as crystalline parasporal bodies and fall into three different size classes, 133-145 kDa; 65-67 kDa and 27 kDa. Different subspecies of Bacillus thuringiensis may express one or more of each size classes.
The 133-145 kDa protein is a protoxin which, when degraded by mid-gastrointestinal proteases, yields an amino terminal fragment of about 67 kDa that contains the toxin moiety (Ogiwara etal, 1992). The 67 kDa toxin shares a significant structural homology with toxins from various other subspecies of Bacillus thuringiensis. The 27 kDa toxin, however, shows no homology with any of the other size classes of toxins but is highly homologous within the subspecies (Luthy et al., 1982). The genes for 27 kDa toxin from subspecies israeliensis (Bacillus thuringiensis israeliensis), kyushunsis (Bacillus thuringiensis kursataki) and morrisoni (Bacillus thuringiensis morrisoni) have been cloned and it has been found that the Bacillus thuringiensis israeliensis toxin differs by a single base from Bacillus thuringiensis morrisoni toxin; whereas it is only 39% identical to Bacillus thuringiensis kursataki toxin (Waalwijk et al., 1985; Ward and Ellar, 1986; Ward et al., 1986; Galjart et al., 1987; Koni and Ellar, 1993). The toxins from various subspecies have been shown to be cytolytic to insect cells in culture.
Almost 20 years ago it was reported that a toxin derived from Bacillus thuringiensis subspecies thuringiensis has antitumor activity against certain murine tumors such as Yoshida ascites sarcoma in vivo. Subsequently, it was shown that a toxin derived from Bacillus thuringiensis israeliensis also has antitumor activity against certain type of murine tumor cells in vitro. The Bacillus thuringiensis thuringiensis protein enhances the humoral immune system in rats and guinea pigs, and induces long lasting antitumor immunity as judged by the rejection of the subsequent tumor transplant. The structural characterstics of this antitumor protein and whether it is functionally related to the insecticidal toxins described above from various other subspecies, is not known.
Murine resident macrophages are poorly phagocytic, exhibit minimal spreading and limited adherence to glass or plastic, and do not possess microbicidal or tumoricidal activity (Wiltrout, et al., 1990). Activated macrophages, in contrast, exhibit increased adherence and spreading, and increased production of mediators of microbial and tumor toxicity such as reactive oxygen intermediates (superoxide, hydrogen peroxide), reactive nitrogen intermediates (nitric oxide), and TNF-.alpha. (Wiltrout, et al., 1990; Green, et al., 1993; and Aggarwal, B. B. 1992). IFN-.gamma. is known to be the major macrophage activating factor; it primes macrophages to react to a second signal such as LPS to optimize the activation process (Wiltrout, et al., 1990; and koerner, et al., 1987). However, full macrophage activation can be also induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) alone (Grabstein, et al., 1986), or by combinations of IFN-.gamma. and TNP-.alpha. or IFN-.gamma. plus IL-2 (Cox, et al., 1992), in the absence of LPS.
The prior art is deficient in the lack of effective means of inhibiting the growth of a wide variety of tumors and for activating macrophages. The present invention fulfills this longstanding need and desire in the art.